poly(A) messages; lost in translation

From a virus' perspective, how do you translate your own messenger RNA (mRNA), whilst not allowing your host cell to continue manufacturing its own proteins, including those that might be detrimental to virus survival? It's a problem viruses have found various ways to overcome, often by manipulating the biology of the mRNAs, which have the following structure:

The classical polyadenylated mRNA ready for translation

Simply, an eIF4F cap-binding complex binds to the cap and a poly(A) binding protein (PABP) interacts with the poly(A) tail. The PABP in turn interacts with eIF4G of the cap binding complex, thus circularising the mRNA for  efficient translation to occur.

The translation complex showing circularisation enabled by PABP linking the poly(A) tail to eIF4G.

A good way in which to specifically translate viral messenger RNAs is to  make the viral mRNAs different in such a way that viral mRNAs are the most efficiently translated mRNAs. Picronaviruses (e.g. polio, foot and mouth disease) and flaviviruses (e.g. West Nile, Hepatitis C), genomes contain an internal ribosome entry site (IRES), which allows ribosome attachment and subsequent translation in the absence of the 5' cap; get rid of the ability of the cell to translate capped mRNAs and suddenly the viral mRNAs are preferentially translated.

Rotaviruses target the other end. Rotavirus mRNAs all end in a specific sequence ....GACC instead of a poly(A) tail. One of the viral non-structural proteins, NSP3, has been shown to act in similar fashion to PABP; NSP3 specifically binds RNAs ending in this sequence (i.e. rotaviral mRNAs), and also binds the cap-binding complex in place of PABP, but with higher affinity than PABP. The overall result is that polyadenylated mRNAs are outcompeted by rotaviral mRNAs. NSP3 also seems to be responsible for PABP accumulating in the nucleus, where it is unable to translate cytoplasmic mRNAs. Even so, there is evidence that rotaviral mRNA translation appears to be independent of NSP3.

A paper has just come out looking into the location of poly(A), i.e. cellular, mRNAs in rotavirus infected cells.
The first question was, where do you find poly(A) mRNAs in infected cells? Using fluorescence in situ hybridisation (FISH) the authors found poly(A) containing mRNAs to accumulate in the nucleus of cells, thus preventing their translation. Removing NSP3 using RNA silencing prevented this from happening, so that poly(A) mRNAs were then found in the cytoplasm, just as in uninfected cells.

Rotavirus (and NSP3) retain polyadenylated mRNA in the nucleus. Oligo (dT) probes detects polyadenylated (i.e. cell-like) mRNA. Normally the mRNA is distributed throughout the cell (top) in infected cells, signal is only observed in the nucleus (middle row) whereas when NSP3 is silenced, the nuclear block is apparently released (bottom).

Next there is an intriguing finding that, perhaps surprisingly, the untranslated regions (UTRs) of rotaviral mRNAs do not influence how well that particular transcript is translated, as luciferase reporter RNAs with host-cell UTRs were not translated any less efficiently than reporter RNAs with rotaviral UTRs. Most strikingly though, was the fact that the overall efficiency of translation appeared to be enhanced in infected cells, implying that the translation machinery is altered upon infection. These mRNAs were directly transfected into the cytoplasm, which isn't how cellular mRNAs originate. To look at this, the authors used two ways of supplying mRNA to the translation machinery, either directly into the cytoplasm, where they again found it to be more efficiently translated in infected cells, or allowed the cell to transcribe it from a plasmid, in which case they observed a decrease in expression as as result of rotavirus infection. Silencing of NSP3 released this apparent inhibition, with the infected cells appearing more like uninfected cells. Together this all leads to the conclusion that, rather than NSP3 affecting the translation of mRNAs directly, the inhibition of poly(A)-dependent translation is due to a lack of export of newly transcribed RNA.

RNA translation in the presence of Rotavirus: (A) when RNA is  transcribed in the nucleus (and needs to be exported for translation), infection suppresses translation, whereas mRNA transfected into cytoplasm implies that rotavirus infection enhances the translation efficiency of the translational machinery. (B) Suppression of rotavirus NSP3 by RNA silencing removes the block imposed upon protein expression from a plasmid.

The authors checked the location of cellular mRNAs too, and found that they too accumulated in the nucleus of infected cells, whereas in mock-infected cells the mRNAs were found in the cytoplasm. Again, when NSP3 was silenced, this block disappeared.

What happens to the polyadenylated mRNAs which accumulate in the nucleus (alongside the normally cytoplasmic PABP which is also retained in the nucleus)? By looking at the length of the RNAs, and using oligos which target the poly(A) tail, they found that the poly(A) tails were increased in length; an observation in line with data showing that PABP accumulation in the nucleus results in hyperadenylation and nuclear retention of RNAs.

Finally, the authors looked to see whether there were more cellular mRNAs in the nucleus compared to the cytoplasm in infected cells. They found that the cytoplasm of infected cells contained 50% less polyadenylated mRNAs. All of this leads to a scenario in which a translationally very active cytoplasm is (comparatively) free of cellular, polyadenylated mRNAs, into which the virus transcribes masses of its own mRNAs; essentially the viral mRNAs now have the cell's translational machinery to themselves, and all of this apparently orchestrated by NSP3.

As a strategy it makes sense; simply get rid of the host's RNAs.

 Rubio, R., Mora, S., Romero, P., Arias, C., & Lopez, S. (2013). Rotavirus Prevents the Expression of Host Responses by Blocking the Nucleocytoplasmic Transport of Polyadenylated mRNAs Journal of Virology, 87 (11), 6336-6345 DOI: 10.1128/​JVI.00361-13
Piron, M. (1998). Rotavirus RNA-binding protein NSP3 interacts with eIF4GI and evicts the poly(A) binding protein from eIF4F The EMBO Journal, 17 (19), 5811-5821 DOI: 10.1093/emboj/17.19.5811

Gold Rush: what the Hoffman crew face

In the four weeks recuperating with a sling from shoulder surgery there's only so much reading and one-handed typing you can do; enter Gold Rush. I found out about this from a research fellow who's arrived from New York. For anyone not familiar (being British I clearly wasn't), this is a reality TV show on the Discovery  Channel following a team of guys from Oregon who pursue the American dream by heading to Alaska aiming to mine gold, with a slight obsession for a 'glory hole'. Inspirational stuff.

Todd Hoffman (centre) and his team head to Alasksa

At one point, Todd and his crew in Alaska seemed to be having issues with mosquitoes, and these could potentially carry arboviruses, (if there were any there to begin with). The water source was rightly another point of concern; it's clearly not just pure virgin meltwater up there.

Gold mining's a dangerous business: as well as the machinery, Alaska also has plenty of inquisitive bears. There's also the added danger of being in the wilderness. One of the prime causes for virus emergence is generally accepted as being encroachment into thus far untouched environments. Disturbing forests or other   

forms of wilderness bring humans into contact with nature, resulting in opportunities for spillover events to occur. As a fairly recent example, and sticking with the mining theme, miners in Uganda experienced an outbreak of Marburg haemorrhagic fever in 2007. In this case just four miners contracted Marburg virus, a very unpleasant virus related to Ebolavirus. As the authors point out though, as long as you go in the cave/mine without protection from the bat secretions (the suspected source of virus), then you'll be at risk. Is the risk worth it? That's going to very much depend on your perspective; a virologist in Scotland is inevitably going to have a different view than someone depending on the mine for their livelihood.

So Todd, if you feel the need to go prospecting in Africa, watch out for those bats.

Coke, formalin, tea...save your horse from African horse sickness!

African horse sickness (AHS) is arguably the most lethal infectious disease of horses. Like Bluetongue virus, AHS virus is an Orbivirus in the Reoviridae family of dsRNA viruses. Also like BTV (and the bunyavirus Schmallenberg virus), it's an arbovirus that is spread between mammalian hosts by Culicoides midges. 

African horse sickness: the lungs fill with fluid and the horse essentially dies  by drowning.  If a horse develops the pulmonary form of the disease, the likelihood is the horse will die.

After recent experiences of BTV and Schmallenberg virus, it's not an unreasonable question to ask whether AHSV would be capable of a similarly large outbreak in Europe. Spain has previously experienced AHSV, but it's never persisted and spread to the extent of the recent BTV and SBV epizootics in Northern Europe.  Though an outbreak is a possibility, BTV and SBV are viruses of ruminants, particularly cattle and sheep, and whilst the midges which feed on cattle and sheep are competent to transmit these viruses, that's no guarantee that similar dynamics would be seen for AHSV. There are also many more sheep and cattle than there are horses, which would also make an AHSV outbreak harder to establish. Nevertheless, the devastating annual experience of AHSV in South Africa suggests that, given the right conditions, an outbreak could happen. Horse-lovers beware.

What can you do? Vaccination? There are both live attenuated and inactivated vaccines for AHSV, but because demand is low most pharmaceutical companies don't formulate them. The most commonly used are the South African live attenuated strains; unfortunately the efficacy isn't great, and if the horses are vaccinated during the midge season they can result in an outbreak.

That leaves therapy. Unsurprisingly people will try anything to save their horse, including some questionable as well the logical approaches (although I'm no pharmacist). I've come across the following, not all of them recommended by a vet:

• Allergex tablets. 
• Vitamin C.
• Tioctan Vet.
• Phosamine or any other Vitamin B Co-injectable.
• Brewers Yeast (can use 2 teaspoons Marmite 3 x per day as a substitute).
• A good probiotic.
• Himalayan Rock Salt.
• Colloidal Silver, a homeopathy classic
• Coca Cola or liquid molasses added to the water to encourage drinking.
• Hydrogen Peroxide 35% in the drinking water of all horses in the yard. 
• DCA immune booster.
• Immune boosting herbs.
• The AHS herbal treatment kit.
• Bute replacement herbs (containing cortisone).
• Eco-Heal, Eco-Lung and Eco-heart.
• Miracle mineral supplement (MMS).
• Salix.
• Dimethyl sulphoxide (DMSO).
• Solal ribose.
• Infrared lamp.
• Oxygen blanket.
• Bio-Electro-Magnetic_Energy_Regulation (BEMER).
• Sub-cutaneous Dettol injections.
• Essential oils (rubbed between the back legs).
• Rooibos tea.

Coke: for AHSV maybe it works, maybe it doesn't, but it's still bad for their teeth. 

The 'cure' I find most concerning though is the intravenous injection of formalin. This seems to be based on historic rumours rather than anything else; one possibility is apparently that it stops the blood vessels from becoming leaky. On the other hand, it's toxic, and I find it surprising that this would even be considered.

Someone with more knowledge of therapies than me please explain, but looking around there doesn't seem to be masses of scientific support for many of these treatments.

At the end of the day though, I really don't blame the owners. If your horse is infected with a virus which is going to kill >90% of those it infects, surely anything's worth a shot!

Schmallenberg Virus RNA in Culicoides midges

There have generally been two views regarding the use of RT-PCR as a way to say whether Culicoides midges are vectors for arboviruses. One side argues that, if you find viral RNA, then the midges are competent. In both the bluetongue virus (BTV) and Schmallenberg virus (SBV) outbreaks in Northern Europe there were thousands of midges mashed and tested for the presence of viral RNA. Of course, midges were found containing RNA. The other side historically replies with something along the lines of, whilst a good indication, this is not evidence of virus replication and spread to the salivary glands, a necessity for transmission to a naive host during the next blood-feed. The hesitation in the latter camp is on the basis that, if you test the entire midge for viral RNA, then there is no way to determine whether any RNA detected is a result of replication, or whether it merely represents virus taken in during the original blood-meal, i.e. it is possible for a midge to be positive, without virus replication taking place.

This all relates to the very first post I wrote - having a sound understanding about what you are detecting and what can be concluded from the result. In this case, it's the fact that RNA does not equate to live virus.

There are ways to gain confidence that RNA presence = competent midge. The simplest is to just argue that the level of RNA is too high to just be a blood-meal, indicating that replication has occurred; this was the case in a recent paper regarding SBV positive midges in Denmark. In this study they also gained confidence that there was viral replication by testing for host (i.e. cow or sheep) RNA; the absence of host RNA implies that the blood-meal had been digested, and therefore the presence of SBV RNA is as a result of virus replication.

Another way is to only test the heads, as an indication that the salivary glands have been infected (a necessity for transmission). The blood-meal is in the abdomen, so any RNA detected in the head will be as a result of replicated and disseminated virus. Dissecting individual midges is a huge undertaking though, and is largely going to be impractical.

In this study by Veronesi et al, midges were either injected with virus, or allowed to feed on blood spiked with virus. After 10 days incubation to allow the virus to replicate and spread, the surviving midges were homogenised and tested by real-time 'semi-quantitative' RT-PCR for the presence of viral RNA. Quantitative RT-PCR would obviously have been preferable, but Cq values (lower value = more RNA) at least give an indication of how intense the infection is.

(A) in the figure above represents RNA levels in midges which had been injected with virus, and shows that RNA was present in the heads and even saliva (8/10 midges tested) of some midges, which may indicate that these midges would be competent to transmit the virus. The lowest values were found in the abdomen/thorax (where the virus was injected), indicating that either the replication was local or the assay was detecting the blood-meal. (B) and (C) are more interesting as they represent lines of midges that are either competent (B, C. sonorensis), or incompetent (C, C. nebeculosus) for BTV, that have been allowed to feed on blood containing virus, thus imitating more closely the 'natural' situation. The C. sonorensis infections resulted in a conspicuous bimodal distribution of Cq values, something which allows the midges to be divided into either transmissible (low Cq values) or non-transmissible (high Cq values) infections. For C. nubeculosus, this distribution was absent, indicating that this species of midge is, like for other arboviruses, non-competent.

Working out what copy numbers of RNA will equate to an infectious midge will be the next step, and this will require the adoption of quantitative RT-PCR.

Culicoides midges have proved themselves to be important vectors of arboviruses, most famously in recent years bluetongue and schmallenberg. The Pirbright lab, originally driven by the great Professor Philip Mellor, have done much work towards unpicking the precise role midges play in virus transmission, particularly in a European situation. Now Philip's one time understudy, Simon Carpenter, and his team are taking things forward, as he explains in the following video about Bluetongue virus.

For a paper looking to implicate midges as vectors of Schmallenberg though, there does seem to be something rather obvious that's missing. Pirbright are in the rare situation of having competent colonies of Culicoides - why not do the key experiment of attempting to infect sheep or cattle with blood-fed midges? RNA, or indeed virus, may be in the saliva in the midges tested for this paper, but that's not evidence that it would be sufficiently infectious to initiate infection in an animal (although the likelihood is it would). 

Overall though, the paper has a slightly different focus - more about the idea of whether or not real-time RT-PCR can be used to indicate whether or not midges are competent vectors. A study tackling this shady area of RNA = infection has needed to be done for a long time; finally it has.


Rasmussen, L., Kristensen, B., Kirkeby, C., Rasmussen, T., Belsham, G., Bødker, R., & Bøtner, A. (2012). Culicoids as Vectors of Schmallenberg Virus Emerging Infectious Diseases, 18 (7) DOI: 10.3201/eid1807.120385
Veronesi, E., Henstock, M., Gubbins, S., Batten, C., Manley, R., Barber, J., Hoffmann, B., Beer, M., Attoui, H., Mertens, P., & Carpenter, S. (2013). Implicating Culicoides Biting Midges as Vectors of Schmallenberg Virus Using Semi-Quantitative RT-PCR PLoS ONE, 8 (3) DOI: 10.1371/journal.pone.0057747

Make me a virus: goodbye MTAs

Material transfer agreements - MTAs - can be infuriating. They will almost certainly exist forever, in some form or other. They say knowledge is power, so it's no surprise that people/institutions want to hold on to anything which may offer them a competitive advantage. It clearly makes sense. I've always been curious though about the amount of metaphorical wheel re-inventing which goes on across the world purely due to the fact that MTAs get in the way; considering the time and money it seems crazy.

Problems arise in the form of time delays and restrictions on what can be done with the materials, whatever they may be. Swapping the simplest of items can take what seems like forever to change hands, and even when it does eventually happen there seems to be a lot of hand tying involved.

But, the concept of virus rescue/reverse genetics means that, for a lot of viruses, the days of the MTA may be limited. Classically, cDNA clones of virus genomes were generated by cloning bits of a virus you already have. This could be extremely laborious and time consuming and meant that you were required to already possess the virus. Now though, gene synthesis is becoming cheaper and cheaper and that means, assuming the sequence is known, that infectious clones can be ordered online by labs with smaller and smaller budgets. Our lab did this recently with Schmallenberg virus. In order to get working as quickly as possible, and allow us to work on whatever aspect we wanted, we ordered the clones online and received them a few weeks later. Not long after, we had Schmallenberg virus, with no restrictions on what it could be used for. 

The virus rescue strategy for Schmallenberg virus; the pUC plasmids were simply ordered online, from Varela et al 2013

For some groups of viruses, rescue systems haven't been established, for example the rotaviruses and some other members of the Reoviridae. For the majority though, there are established systems and clones, including many viruses which are under strict restrictions in laboratories. Accession number AF086833 is the full genome sequence of Ebola Zaire 1976, the original strain associated with horrifying levels of mortality. Similarly, accession number AJ539141 is the sequence of the Foot and Mouth Disease Virus from the UK in 2001 which was estimated to cost the UK government £8bn ($16bn). I'm not aware of questions being asked when ordering such sequences to be synthesised. I thought about experimenting and doing some sort of dummy orders of sequences such as these online to see whether there were any blocks in the way. In theory there should be questions from the company regarding what it is and where it's being sent once it's made. All I know for now is that we were essentially able to order a newly emerged virus online. But arguably the most positive aspect of this, is that it hasn't taken months of paperwork to formulate a restrictive MTA!

Man flu or real flu? - DIY Diagostics

Someone in the lab emailed me today saying that she wouldn't be coming in as she was ill, and that she hoped it was a cold instead of flu. If you're in the same situation, wouldn't it be good if you could test yourself to find out? Well, you can, using a simple dip-stick style test which you can buy online.

That's great and it's easy to envisage how useful this can be but, for me, the interesting page is the one describing the test specifications - how good is the test? The most notable values are the positive (PPV) and negative (NPV) predictive values, which represent the accuracy of a result; PPV represents the proportion of positive results that are truly positive and likewise the NPV is the proportion of test negatives that are truly negative. A PPV of 62% for a nasal swab doesn't seem to be particularly high. As these values very much rely upon seroprevalence it's hard to really tell how useful this is. 

These assays do work, and simple lateral flow devices worked really well during the foot and mouth outbreak in 2007 in the UK. These assays currently use antibodies and, without a good antibody, assay sensitivity can be poor. In lab settings, molecular assays based upon detecting viral nucleic acid are generally more sensitive, in particular real-time RT-PCR. Sadly, molecular methods also require much more expensive equipment, making it more difficult to convert a lab procedure into a 'point-of-care' test. Perhaps unsurprisingly, the initial major steps were taken by the military as a response to the threat of biowarfare. Now however, more commercial machines have made their way to the market, for example machines made by Smiths detection.

Smiths detection PCR machine: essentially a robot for nucleic acid extraction and a real-time PCR machine packaged in a (very heavy and expensive) briefcase.

Field based diagnosis is as close now as it has ever been. Ultimately though, the problems of hardware still exist. The machines are expensive; essentially they are the same machines that a lab has packaged into a briefcase. I think molecular assays in the field will only really take off once isothermal assays, such as loop-mediated isothermal amplification (LAMP), are more widespread. As their name suggests, isothermal assays are performed at a single temperature, so all that is required is a simple waterbath set to a particular temperature, as opposed to a block of metal heating up and cooling down. 

Loop mediated isothermal amplification: modified primers loop back to prime the alternative strand. A strand displacing enzyme abrogates the need for variable temperatures.

Detection is the other problem; fluorescence as a read-out is expensive to a) achieve and b) detect, therefore the gold standard for field-based detection of amplification (PCR, LAMP etc.) is likely to be dipsticks/lateral flow devices as these are easy, more foolproof and provide a clear answer. For now, extraction of the nucleic acid probably remains the biggest obstacle. Another benefit of assays based around isothermal amplification and simple methods of detection is that it allows the use of molecular assays with minimal equipment, meaning that such assays can then be used in resource poor countries. 

It is more than likely that the future will be much more sophisticated with much greater scope for what can be detected, although the requirements for simplicity are unlikely to change much. For now, imagine settings such as clinics where patients suspected of hepatitis C can get an instant result. LAMP assays are already available for many of the pathogens that would be on a list of desirable tests; I suspect it won't be long before point of care testing becomes even more widespread than it already is.

Lessons from Schmallenberg

Whilst I haven't spoken to one, being a UK sheep farmer at the moment can't be much fun. Schmallenberg virus (SBV) is robbing farmers of lambs as well as being an altogether disturbing and unpleasant experience. How long it remains an issue will only become apparent with time. It's worth looking at whether this outbreak of SBV represents a sign of what's to come.

The Hollywood films all seem to make a big issue of a virus 'going airborne'. That's unsurprising considering airbrone spread is probably one of, if not the, most efficient form of transmission when it comes to humans and their viruses, crammed together in offices and public transport. I'm not so sure it's the same for livestock viruses though, where the densities of animals may be locally high (i.e. within a farm), but with greater spaces between groups (i.e. between farms). Even though Foot and Mouth disease virus is airborne, and pig farms in particular can release sufficient amounts of virus into the air to infect other properties, the majority of the FMDV spread in 2001 in the UK was due to both direct contact between animals and indirect contact through so-called fomites. Arboviruses, on the other hand, are transmitted when insects find and bite animals, resulting in a form of transmission which is more heat seeking missile than carpet bomb. Both of the most recent two exotic livestock viruses to enter the UK (Bluetongue and Schmallenberg) have been spread by insects, more specifically, Culicoides midges. The fact that it's midge-borne viruses that have made it to the UK may be as a result of the midges being small enough to be blown in wind plumes, including across the channel from the continent where the viruses emerged. 

Wind plumes blowing across to the UK from the continent; the probable route of entry
into the UK for SBV (and a few years earlier Bluetongue virus).

Whilst wind may explain why midge-borne viruses reach the UK, it doesn't explain why it's midge borne viruses that have spread across the continent so fast. Why aren't mosquito-borne viruses going just as crazy? West Nile managed to spread from the US east coast to the west in just a few years.

Image from CDC.

Could it be insect vectors provide an efficient form of virus transmission providing the climate is right? It certainly seems so once it's introduced, although maybe not ideal for global spread when considering the relative immobility of livestock compared to humans. How Schmallenberg (or Bluetongue) arrived in the Europe to begin with is open to speculation; but the fact that it spread extremely fast across Northern Europe is undeniable. Within months of the initial observations in Germany, SBV had made its way to the UK and, within one summer, had more or less spread throughout the entire country.

The rapid spread of SBV; from covering North West Germany, Belgium and the Netherlands in the spring (top, 5th Jan 2012) to large swathes of Europe, including the UK, by the autumn (bottom, 26th October 2012).

And the future? Just like events such as the spread of West Nile virus in North America, we should perhaps regard it as a warning. Rift Valley fever seems to be the vogue virus in terms of a likely prospect for the future in Europe. Rift Valley, West Nile, as well as the majority of other candidates such as Chikungunya virus are all spread by mosquitoes; what about other Culicoides- borne viruses? First, African horse sickness; the most lethal infectious disease of horses, with up to 90% mortality, is a close relative of Bluetongue, as is epizootic haemorrhagic disease virus which, whilst still only on the fringes of Europe, already causes problems in the US. Human viruses? Oropouche virus (like SBV, an orthobunyavirus) can cause a dengue-like illness. Oropouche currently seems to be limited to South America; not many people would right now put a lot of money on it making its way to the UK. Before 2006 though, nobody thought Bluetongue virus would seriously enter Northern Europe, let alone go on to infect the vast majority of sheep and cattle in the region. Uncertain times are ahead.